Research shall be continued or initiated in the following areas: (1) On the properties and mechanism of action of 2-keto-4-hydroxyglutarate aldolase, an important enzyme in the metabolism of L-hydroxyproline by mammals which has been found to differ in many respects from other aldolases studied so far. We have the enzyme in highly purified form from extracts of bovine liver and are attempting to purify it from bovine kidney. This aldolase is also present in E. coli; homogeneous samples of the enzyme are routinely prepared from this source. Our efforts include an examination of the subunit structure of the enzyme, an exploration of the function groups in the enzyme molecule which participate in the catalytic process, isolation of active-site peptides, and the establishment of structure-function relationships in general. Comparative enzymology is done. (2) On the nature of new reactions and enzymes in the metabolism of gamma-hydroxyglutamic acid, an intermediate in L-hydroxyproline degradation by mammals. Special attention is now being given to delineating the nature of the reaction wherein 2-keto-4-hydroxy-glutarate is oxidized to malate plus CO2 and to establishing the physiological role of this conversion. (3) On the purification, properties, and role of L-threonine dehydrogenase and D-1-amino-2-propanol: NAD oxidoreductase, enzymes found in animals and bacteria that are involved in the catabolism of L-threonine. These two enzymes also have the potential role of providing the D-1-amino-2-propanol moiety utilized in vitamin B12 biosynthesis by bacteria.